A range of strategies have been developed for the high throughput quantitative/comparative analysis of large scale systems to enable the expression levels of various molecules to be compared between different biological states. In the—omics fields, the techniques used in the field of proteomics are probably the most developed, and can be broadly subdivided into two general schemes—those that involve the use of labels and those methods that are label free. In the label free approaches, various aspects of the peptides/proteins such as normalized ion intensities, spectral counts, mass, scan number and signal intensity, and accurate mass plus retention time have been successfully used to assign the protein expression level for comparative investigations1-4. However, the ionization efficiency of each analyte depends on factors such as its molecular mass, proton/cation affinity, surface activity, the presence of other compounds which compete with or interfere with the ionization of the analyte, etc., and thus the intensities of the ions do not directly correlate with concentration. In addition, the instrument's response can vary over time, so that the direct comparison of data from two or more analyses can yield dramatically different results. The alternative strategy for relative quantification overcomes these issues by simultaneously analyzing pairs of isotopically labeled populations, which offers the advantage that the heavy and light labeled peptide pairs are analyzed under exactly the same conditions, allowing a direct comparison of relative abundances for that peptide. Relative quantification between the isotopically labeled populations is performed by taking a ratio of the area or the intensity of the light and heavy monoisotopic peaks.
Numerous strategies have been developed for introduction of a stable isotope into populations of proteins5-12. For example, isotope-coded affinity tags (ICAT) chemically target specific amino acids, typically cysteine, in the peptide sequence for differential labeling5. Other in vitro approaches also target functional groups of the polypeptides6, 8-12. Stable isotopes can also be introduced into biological systems via metabolic labeling. For instance, stable isotope labeling with amino acids in cell culture (SILAC) provides a simple and straightforward method for the in vivo incorporation of an isotopic label into proteins prior to MS based proteomics7. In a SILAC experiment, two cell populations are grown in culture media that are identical except that one of them contains a “light” and the other “heavy” forms of particular amino acids (12C and 13C labeled lysine and arginine for example). The labeled analogs of amino acids are supplied to cells in culture instead of the natural amino acids, and it becomes incorporated into all newly synthesized proteins. After a number of cell divisions, each instance of the particular amino acids is replaced by its isotope labeled analog. An advantage of this approach, over the in vitro approaches, is that the cells are mixed together immediately after cell lysis. Thus, proteins from both cell types are subjected to the exact same experimental conditions during sample handling, digestion, purification, etc., which eliminates the differential losses that can occur when the samples are treated separately in a parallel manner. For this reason, SILAC is often considered the “gold standard” for quantitative proteomic analyses13.
The field of comparative glycomics is not as mature as proteomics; however several of the quantitative proteomic tools have been adapted for glycomic analysis. For instance, total ion mapping (TIM) is a label free method that determines the prevalence, percent of an individual glycan to the total of all glycans in the sample, based upon the sum of the fragment ions intensities, and is in some ways similar to the label free methods used in proteomics14. In vitro isotopic labeling has also been developed by a number of groups. For N-linked glycans and free oligosaccharides, several isotope containing tags have been derived to label the reducing terminus15-18. O-linked glycans are usually released from the protein backbone via β-elimination, and thus are not amenable to these approaches. However, a quantitative method relying on β-elimination to introduce a mass label into the glycan has been developed19. Another proposed method for comparative isotopic labeling of oligosaccharides relies on heavy methyl iodide (13CH3, 12CDH2, 12CHD2, and/or 12CD3) vs. light methyl iodide (12CH3) labeling during standard permethylation, which is a commonly used derivatization procedure prior to MS analysis of both N-linked and O-linked glycans14, 20, 21. In addition, an isobaric labeling strategy using permethylation with 13CH3 and 12CDH2 has been developed for both N- and O-linked glycans, and is particularly useful for the quantification of individual glycans present in isomeric mixtures22, 23. All of these in vitro approaches are useful tools for glycomics.